1

Formation of biological condensates via phase separation: Characteristics,

analytical methods, and physiological implications

Zhe Feng1, Xudong Chen1, Xiandeng Wu1, and Mingjie Zhang1,2,*

1Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong

Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong,

China

2Center of Systems Biology and Human Health, Hong Kong University of Science

and Technology, Clear Water Bay, Kowloon, Hong Kong, China

Running title: Biological condensates formation via phase separation

* To whom correspondence should be addressed: Mingjie Zhang: Division of Life Science,

State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and

Technology, Clear Water Bay, Kowloon, Hong Kong, China;

mzhang@ust.hk; Tel. (852) 2358 8709.

Keywords: phase separation, biological condensates, protein-protein interaction, cell

signaling, cell biology, cellular regulation, membraneless organelle, intrinsically

disordered protein, scaffold proteins, multivalent interactions

ABSTRACT

Liquid–liquid phase separation (LLPS)

facilitates the formation of condensed

biological assemblies with well-

delineated physical boundaries, but

without lipid membrane barriers. LLPS is

increasingly recognized as a common

mechanism for cells to organize and

maintain different cellular compartments

in addition to classical membrane-

delimited organelles. Membraneless

condensates have many distinct features

that are not present in membrane-

delimited organelles and that are likely

indispensable for the viability and

function of living cells. Malformation of

membraneless condensates is

increasingly linked to human diseases. In

this review, we summarize commonly

used methods to investigate various

forms of LLPS occurring both in 3D

aqueous solution and on 2D membrane

bilayers, such as LLPS condensates

arising from intrinsically disordered

proteins or structured modular protein

domains. We then discuss, in the context

of comparisons with membrane-

delimited organelles, the potential

functional implications of membraneless

condensate formation in cells. We close

by highlighting some challenges in the

field devoted to studying LLPS-mediated

membraneless condensate formation.

INTRODUCTION

In eukaryotic cells, reaction components

are spatiotemporally compartmentalized

such that materials are concentrated,

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activities are localized and protected from

damaging activities such as proteolysis,

changes in pH and undesired covalent

modifications. Classical organelles are

membrane-enclosed where the lipid bilayer

provides a physical barrier to separate their

interior contents from the exterior

environment. Examples include Golgi

apparatus, mitochondria and endoplasmic

reticulum (ER). However, many organelles

are not membrane-enclosed (often referred to

as membraneless compartments in the

literature), and such organelles include but

not limited to germ granules, stress granules,

nucleoli, centrosomes and synapses in

neurons. In these membraneless

compartments, due to the lack of physical

separation, molecules can freely exchange

with their counterparts in the surrounding

bulk solution. Sharp concentration gradients

are maintained between the proteinaceous

(and sometimes protein and nucleic acid

mixtures) interior and the much more diluted

exterior. Reaction machineries can reversibly

assemble and disassemble within a short time

window, as fast as a few seconds. Reaction

constituents can be integrated or removed to

control specific activities. Although

recognized for many years, the mechanisms

governing the formation of membraneless

organelles have remained unclear until about

10 years ago. First direct experimental

evidence came from the study of P granules

in germ cells of Caenorhabditis elegans (1).

P granule is a collection of RNA and RNA

binding proteins (RBPs) localized at the

posterior cortex of a dividing embryo. P

granules appear as spherical droplets with

liquid-like properties− fuse with one another,

deform under shear stress and flow off the

surface of nucleus. Fluorescence recovery

after photobleaching (FRAP) analysis

demonstrated rapid turnover rates of

constituent proteins, which is indicative of

fast molecular rearrangements. These

observations together suggested that P

granules form through liquid-liquid phase

separation (LLPS), distinct from canonical

macromolecular assemblies. Since then the

list of membraneless organelles that are

organized by LLPS has ever been growing.

Nevertheless, early concerns had been raised

over the specificity of phase-separated

condensates observed in vitro and their

biological significance in vivo.

Comprehensive studies were followed to

show that the concept of phase separation can

help to explain the formation and

organization of non-membrane bound

biomolecular compartments as well as their

physical and material properties that cannot

be understood with the classical physical

chemistry theories for dilute solutions. It now

comes to the realization that LLPS might be

a general mechanism to drive

compartmentalization in the absence of lipid

bilayers (2-7), and this has greatly motivated

the field to re-investigate mechanisms

underlying formation and functional

implications of membraneless organelles

from new perspectives. However, cautions

should be exercised that not all condensed

phase properties observed in vitro can be

extrapolated to living cells. Rigorous

characterizations, both in vitro and in vivo,

are required to demonstrate the existence of

LLPS of a particular biological system under

physiological conditions. Here we first

review our understanding about the

molecular codes that contribute to LLPS

formation. We discuss some of the common

techniques for characterization of LLPS. We

then discuss the functional implications of

LLPS-driven organelle assemblies. Finally,

we propose a few new potential research

directions inspired by current works on LLPS.

What is phase separation?

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By definition, phase separation refers to

the immiscibility of two solutions whereby

they separate into two states. In biological

systems, this often leads to a large volume,

dilute liquid phase and a small volume,

concentrated liquid phase. In biology, phase

separation is not unheard of. In protein

crystallization, when a crystallization reagent

is added to the protein solution oil-like

droplets can be observed to separate from the

bulk solution. LLPS in this case is indicative

of a metastable transition state from which

crystals may grow by changing temperature,

precipitant concentration, protein

concentration etc. Molecules are miscible in

solution until reaching their solubility limit.

Phase separation happens when the

macromolecule/macromolecule or

solute/solute interactions are energetically

favored over the macromolecule/solute

interactions and the gain in free energies is

favored over its loss in entropic tendency

towards homogenous solution state (6,8,9). A

free energy minimum is then reached, but the

two phases with different solute

concentrations are at the same Gibbs free

energy (4). For each molecular system, a

phase diagram can be constructed by

systematically screening through conditions

such as temperature, salt concentration, pH

or macromolecular concentration. Phase

diagram helps one to identify conditions that

promote phase separation and to determine

the likelihood of phase separation happening

under physiological conditions (Fig. 1A).

Phase boundary, which is defined by the

binodal line, indicates the boundary that two

distinct phases stably co-exist in solution.

Outside the binodal curve, molecules stay as

homogenous mixtures. Between the binodal

and spinodal curves lies a metastable region

where liquid demixes via a nucleation

process. Within the spinodal zone, spinodal

decomposition occurs. In the other words,

spontaneous phase separation takes place in

the spinodal zone where molecules rapidly

transit from a less stable region to a more

stable phase separated region bypassing the

metastable nucleation zone.

Multivalency is a key determining

factor underlying LLPS in biological systems

(10). Molecules can undergo inter- or intra-

molecular interactions to assemble into

oligomers or polymers which tend to have

lowered solubility limit and thus more likely

to demix with the surrounding solution. In a

folded domain protein, multiple binding sites,

either for itself or for its binding partners,

promote phase separation (Fig. 2). In proteins

with higher content of intrinsic disorder,

multivalent weakly self-attracting

interactions collectively drive phase

separation (Fig. 1B-D). Aggregations of

misfolded cytosolic or nuclear proteins have

been associated with a broad range of

neurodegenerative diseases such as

Alzheimer’s disease (AD), Parkinson’s

disease (PD) and amyotrophic lateral

sclerosis (ALS) (11-13). Solid fibrils formed

by disordered proteins represent another

form of phase separation− sol to solid

transition. Pathological aggregation and its

close link to brain diseases are discussed in

several recent reviews (14-19). For the scope

of this review, we focus on recent works on

LLPS. Below we discuss the sequence

properties coding for LLPS, the biological

functions of condensate formation and the

technical developments for in vitro

characterization of phase separation systems.

Codes for LLPS

Tremendous progresses have been made

over the past decade trying to understand the

molecular features in common that drive

phase separation. In this section, we discuss

examples of phase separation promoted by

intrinsically disordered sequences or more

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specific folded domain/target interactions.

Multivalency driven by intrinsic disordered

sequences

Intrinsically disordered proteins

represent an abundant class of proteins

involved in phase separation. Low

complexity regions (LCRs) show biased

amino acid preferences including Gly, Ser,

Asn, Glu, Phe and Tyr. These amino acids

often appear in repeats such as RG, FG and

YG repeats that are important for forming

ribonuclearprotein (RNP) granules in P

bodies, P granules, stress granules in

cytoplasm or nucleoli and paraspeckles in the

nucleus (20-25). The lack of a defined three-

dimensional (3D) structure in LCRs favors

weakly adhesive interactions that drive phase

separation. A good example studied in detail

is Fused in Sarcoma (FUS) proteins. Full

length FUS proteins have been shown to

undergo LLPS at close to physiological

concentrations in the presence of crowding

reagent or when cooled to 4 °C (26,27). Gel-

like state is observed when FUS protein

concentration reaches hundreds of μM, well

above its physiological concentration. FUS

LCR alone can assemble into hydrogels at

sub-mM concentration (28), and the

hydrogels trap and retain the LC domains of

other RBPs such as hnRNPA1, Sup35, TIA1

and TDP43, although to different levels. The

liquid droplets of FUS protein can further

mature into fibrous aggregates resembling

the pathological protein fibers found in ALS

patients. Mutations in the prion-like domain,

which induce the early onset of ALS, further

promote the sol-solid transition. Tremendous

progresses have been made in recent years in

revealing the emergent sequence

determinants in LCR that promote phase

separation. Noticeably, the types of

interactions critical for phase separation are

commonly known to drive protein folding or

interactions. We discuss below how these

sequence features can drive molecular

interactions in new ways.

Intrinsically disordered proteins rich in

aromatic residues are favored to form pi-pi

stacking interactions that can drive phase

separation (25,28-30) (Fig. 1B). Mutation of

these aromatic residues to serine can strongly

decrease the amount of protein enrichment

into condensed phase. Apart from side chain

pi-pi interactions, small residues with

relatively exposed backbone peptide bonds

can also form the so named planar pi

interactions (31). Gly, Ser, Thr and Pro

residues are indeed frequently found in LCRs

of RBPs. RG/RGG repeats are also found in

multiple LCR containing proteins such as in

the nuage protein DEAD-box helicase 4

(Ddx4) (25), the P granule protein LAF-1 (22)

and the neuronal granule protein Fragile X

Mental Retardation Protein (FMRP) (32,33).

Arginine can form cation-pi interactions with

aromatic residues, either intramolecularly or

intermolecularly (Fig. 1B). Increase in the

number of cation-pi interactions by arginine

substitutions in FUS protein, for example,

can significantly promote its ability to phase

separate and lower the threshold

concentration of the sol-gel transition (30,34).

Conversely, substitution of arginine and

tyrosine or phenylalanine with alanine

disrupts cation-pi interactions and

consequently the ability to phase separate.

Similarly, arginine methylation in Ddx4 (25),

hnRNPA2 (35) and FMRP (33) reduces or

abolishes their phase separation likely

because of the weakened intermolecular

cation-pi interactions. Charged residues also

contribute to droplet formation both in vitro

and in vivo (Fig. 1C). Ddx4, for example,

contains blocks of net negative or positive

charges, typically 8-10 residues in length

with 3-8 charged residues (25). Interestingly,

these charge blocks appear in clusters with

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alternating positive and negative charge

distributions. Removal of such opposite

charge patterning inhibited Ddx4 phase

separation. Apart from amino acid side chain

interactions, phase separation can be driven

by secondary structural elements. Examples

include a short, evolutionarily conserved

helical segment in TDP-43 C-terminal

domain involved in intermolecular helical

interactions (36) and a 57-residue segment in

FUS LCR involved in the formation of cross

β-sheets stabilized by hydrogen bonding and

pi-pi stacking interactions (37). Recent

crystallographic studies of LCRs in FUS,

hnRNPA1 and nup98 revealed another type

of interactions between secondary structural

elements that drive phase separation. These

regions are highly abundant in aromatic

residues, which are involved in inter- and

intra-sheet stabilizations. In addition, the

LCRs form kinked β sheets to allow close

encountering of the backbones for hydrogen

bonding or Van der Waals interactions and

subsequently to stabilize the packing of

neighboring β sheets. Such regions are

therefore referred to as low complexity

aromatic-rich kinked segments (LARKS) (38)

(Fig. 1D).

Based on current knowledge of

relationship between emerging sequence

features and phase separation, a spectrum of

predictive tools has been developed to enable

researchers to identify regions in intrinsically

disordered proteins that might be involved in

LLPS and to understand the molecular

mechanisms behind sol-sol/gel transitions.

This has been extensively discussed in a

review written by Alberti and colleagues (3)

Multivalent interactions driven by defined

modular protein domains

Experiments to manipulate the valency of a

folded protein have proven an inverse

correlation between the number of binding

domains or motifs and the saturation

concentration above which the system

undergoes phase separation. For instance,

repeats of SH3 domain bind Pro-rich motifs

(PRMs) and phase separate into condensed

droplets upon mixture at high concentration

(10). The phase boundary (i.e. the threshold

concentration for LLPS) is lowered when the

number of binding modules increases

suggesting that LLPS is strongly dependent

on the valency of interactions. Similarly,

multivalent nucleic acid/protein interaction

systems are known to undergo LLPS both in

vitro and in cells when certain critical

numbers of valency are reached (39,40).

There are now many examples of phase

separation systems driven by modular

domain interactions. One example is the

multivalent protein network involving the

transmembrane protein nephrin, the adaptor

protein NCK and its ligand N-WASP that

regulate actin assembly in podocytes of

kidney (10,41) (Fig. 2A). NCK contains three

SH3 domains, each of which can bind to the

six PRMs in N-WASP; two proteins

assemble into higher order oligomers that

phase separate. This process is accelerated by

nephrin addition where phosphor-tyrosine

(pTyr) residues in nephrin bind to SH2

domains in NCK. The assembled droplets

can further recruit Arp2/3 complexes for

actin polymerization. An analogous system is

observed in T cell receptor signaling (42).

LAT, a transmembrane protein for T cell

activation, is phosphorylated at multiple Tyr

sites that are required for T cell signaling.

Addition of Grb2, an adaptor protein, and

Sos1, a guanine nucleotide exchange factor

for Ras GTPase, caused phase separation of

pLAT through interaction between pTyr in

LAT and SH2 domain in Grb2 and between

SH3 domains in Grb2 and PRMs in Sos1.

The number of pTyr residues affects binding

valency in the system and consequently the

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efficiency of receptor clustering on supported

lipid bilayer. In both systems, properties of

reconstituted condensates in vitro strongly

correlate with the observations in living cells.

In neurons, synapses assemble between

axons and dendrites. Beneath the

postsynaptic membranes there lies an

electron dense layer of material known as the

postsynaptic density (PSD). Addition of

SynGAP, a negative PSD activity regulator,

to the major PSD scaffold protein PSD-95

caused droplet formation (43). Phase

separation was completely abrogated if the

interaction interface or SynGAP trimer

interface was impaired. PSD-95 also

clustered with SAPAP, Shank and Homer,

which are major PSD scaffold proteins, but

with much higher efficiency compared to

SynGAP (44) (Fig. 2B). This increased

propensity to form droplets is likely because

of increasing valency provided by

multivalent interaction interfaces among

PSD constituents. Importantly, all of the

interactions involved in forming the PSD

protein network are highly specific and with

strong affinities, and these interactions

involve well folded protein binding domains

(Fig. 2B). Strikingly, the assembly of PSD

droplets was dispersed when Homer1a, a

monomeric splice variant of Homer1, was

added to the pre-assembled condensates (44).

This suggests Homer1 oligomerization plays

a crucial role in promoting LLPS.

Reconstituted PSD condensates can further

cluster the cytoplasmic tail of NMDA

receptor subunit and nucleate actin

polymerization both in solution and on

supported lipid bilayer.

The presynaptic active zone is also

organized by phase separation (45). As

viewed under the electron microscope, active

zone comprises densely packed proteins,

which organize into a layer of electron dense

projection beneath the presynaptic

membranes. RIM and RIM-binding protein,

two major active zone scaffold proteins,

formed liquid droplets upon mixing. RIM-

binding protein contains three SH3 domains,

each of which binds to Pro-rich motifs in

RIM (Fig. 2C). The cytoplasmic tail of

voltage-gated Ca2+ channel (NCav) is also

enriched into RIM/RIM-binding protein

condensates, and this co-clustering

significantly lowers the threshold

concentration to undergo LLPS. When NCav

C-terminal tail is attached to membrane, RIM,

RIM-binding protein and NCav co-cluster on

supported lipid bilayer, providing a

mechanistic explanation to the tight coupling

of Ca2+ influx and neurotransmitter release in

presynaptic termini.

In addition to these systems,

aggregation of Rubisco by the protein CcmM

(46) and interaction between the tetravalent

RNA binding protein PTB and an RNA

oligonucleotide (10,47) are also shown to

phase separate through multivalent folded

domain interactions. In RBPs, phase

separation can be driven by modular domain

interactions apart from those weak, self-

adhesive interactions. In particular, RGG

repeats and RNA recognition motifs (RRMs)

are involved in RNA binding. Although

binding between individual repeats and RNA

is relatively weak, multiple RGG repeats

together generate a high affinity interaction

and crosslink proteins and RNAs into higher

order oligomers (33,36,48-50). Repeats of

RRMs on RBPs and multiple RRM binding

sites on RNA provide further degree of

multivalency to promote phase separation.

Addition of RNA to hnRNPA1 caused

formation of liquid droplets (24). In other

cases, RNA is readily recruited to the pre-

assembled liquid droplets or hydrogels via

charge-charge interactions (33,51-54). It

appears that intrinsically disordered regions

and high affinity domain interactions can

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both contribute to phase separation. In reality,

it is likely that both types of multivalency are

coupled to promote droplet formation in

many systems.

Methods for observation of phase

separation

When it comes to work with phase

separation in vitro, multiple approaches are

generally combined to describe the

phenomenon of phase separation, to

distinguish it from non-physiologically

relevant aggregations or simple binding-

induced molecular assemblies, and probably

most importantly, to link to its biological

functions in cells.

3D solution system

The most intuitive observation of phase

separation in a test tube may be the turbidity

and opalescence of a solution when

components are mixed under certain

conditions (protein concentration,

stoichiometry, salt concentration, pH,

temperature etc.). Such simple sedimentation

assay can be used to quantitatively evaluate

the fractional distribution of proteins in each

phase (see (43) for an example). A

transparent “pellet” observed after

centrifugation implies a liquid phase rather

than aggregates or precipitates. Alternatively,

direct measurement of the turbidity (46,55,56)

is also helpful for estimating the extent of

phase separation. But practically, due to the

non-negligible gravity of the condensed

phase droplets, researchers should be

cautious about the heterogeneity within final

solution, and two experimental setups must

be considered: 1, a vertical, instead of

horizontal, light beam is recommended for

absorbance measurement to gain a more

reliable result. 2, sample solution needs to be

vortexed immediately before the

measurement to ensure a homogenous

suspension.

Imaging assays are necessary to confirm

the liquid-like properties of condensed phase.

Differential interference contrast (DIC)

imaging is the most straightforward method

to depict the coexistence of two (or more)

distinctive phases (Fig. 3A). The spherical

morphology, fusion upon contact, and droplet

fission, as well as the deformation of droplets

under shear forces, together demonstrate the

liquid-like properties of condensed phase

(1,4,6) (Fig. 3B-D). Combining with

fluorophore labeling, either colocalization or

coexistence of sub-compartments can be

visualized (57) (Fig. 3A). But we should

always be alerted of potential imaging

artifacts and should not merely rely on

imaging assays for the following reasons: 1,

cross-talk between multiple channels is not

easy to be completely blocked, and it will

always give an image when high laser power

and long exposure time are applied. Thus, we

recommend using single fluorophore

labeling, if possible, in a given system except

for co-localization or other necessary

conditions. 2, the conjugation of a

fluorophore (or genetically encoded

fluorescent tag) may affect the properties of

labeled protein, and relatively subtle impact

may be augmented in a much more

concentrated phase. In addition,

overexposure of high percentage labeled

protein may cause photobleaching of the

fluorophore under continuous laser power,

therefore we recommend researchers to

dilute the labeled protein with unlabeled one

to achieve sparse labeling (usually, ~1% is

sufficient). It is helpful to use sedimentation

assay with labeled protein to check the

potential effect of labeling on its ability to

phase separate. 3, it’s hard to accurately

judge whether a protein of interest is

specifically retained in the condensed phase,

especially when the fluorescence contrast to

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the surrounding environment is not high

enough. Since the matrix pore size (i.e. the

void space between the protein network mesh)

of condensed phase is deemed to be large

enough to accommodate normal protein, as

indicated by our recent observation that the

dodecameric CaMKII (~550 kDa) can

penetrate the reconstituted condensed PSD

phase in vitro (44), it is not surprising that

even some irrelevant proteins can go through

but do not prefer the condensed phase.

Nonetheless, fluorescent imaging can

provide valuable information with rigorous

controls. Recently, we developed an absolute

concentration estimation method based on

measured fluorescence intensity (44) (Fig.

3E). A standard curve is first constructed by

plotting the fluorescence of protein measured

at known concentrations. Based on this curve

we can then back calculate the exact

concentration of components in a condensed

phase, even though the concentration in the

surrounding dilute phase cannot be precisely

estimated due to the very low signal to noise

ratio. Protein concentration in the dilute

phase may be determined taking into account

of its fractional distribution observed in

sedimentation assay. Concentration ratio and

therefore, volume ratio between condensed

and dilute phases can ultimately be estimated

(45).

FRAP analysis is increasingly adopted

to demonstrate the mobility and dynamics of

molecules within liquid droplets (Fig. 3C).

Molecules exchange within condensed phase

(half-bleach) or exchange between

condensed and diluted phases (half-/whole-

bleach) can be faithfully captured by FRAP

experiments. However, we should be

cautious in assessing the fitting of

fluorescence recovery curves because it can

always give us a result regardless of whether

the model is appropriate or not. Fluorescence

recovery is dependent on the movement rates

of molecules, but this “movement” consists

of diffusion (in the dilute phase, condensed

phase, and the interface) and interaction

(dissociation koff and association kon). It is

therefore difficult to derive an exact value of

either characteristic diffusion rate constant (τ)

or diffusion coefficient (D) before figuring

out a theoretical model, even though the

apparent τ and D provide certain referential

meanings. Besides, a less mobile or

immobile fraction may occur, and a second

bleach immediately after the plateau of the

first bleach is suggested to confirm this. The

immobile fraction can be generated from

systematic background which is an intrinsic

property or produced during imaging time by

rapid hardening/ageing. In some systems, the

phase droplets are initially fluid, but their

viscoelasticity increases over the time and

molecules eventually cannot exchange with

their counterparts in the surrounding solution.

This process is known as hardening/ageing,

although the mechanism behind it is

currently unknown.

Apart from direct visualization, several

techniques have recently been brought into

phase separation field to describe the

material properties of biological condensates.

The isolated droplets make it possible to

monitor the material properties of individual

phase via atomic force microscopy (AFM),

from which the stiffness, viscosity, elasticity,

pore size, and other soft material parameters

can be quantitatively extracted. This

information will provide useful insights into

the behavior of biological condensates in

cells. For example, AFM measurements of

the mechanical properties of PSD droplets

indicate that the 6-component PSD

condensates is more gel-like comparing to

the 2-component PSD condensates

reconstituted in vitro (44). It is thus

reasonable to speculate that under

physiological conditions, PSD may be a more

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gel-like structure due to the more complex

valences and more crowded environment,

which fits well to the previous electron

microscopy (EM) observations (58).

Measured material properties also provide

explanations towards the observed protein

dynamics in vitro. The time-dependent

hardening indicated by elastic modulus

values suggested an aging process of

reconstituted PSD condensates, consistent

with the observation that PSD constituents

demonstrated a time-dependent decreasing of

the signal recovery in FRAP analysis (44).

How are molecules organized within the

condensed phase? Researchers show great

interests towards understanding the atomic

details of condensed phase. The intrinsic

heterogeneity, highly dynamic properties,

and the numerous transient interactions

existing in liquid-like phase make it

extremely difficult to obtain structural

information (59). Nevertheless, protein

concentrations within condensed phase

remain the same in a given condition (pH, T,

salt etc.) (3,44), bringing hopes to acquire

some configuration rules from structural

studies. A recent study combing cryo-EM

and cryo-electron tomography (cryo-ET) to

solve the structure of Rubisco-CcmM

complex under LLPS condition may give us

some inspiration (46). Cryo-ET analysis of

clusters of Rubisco complexes revealed that

the median nearest-neighbor distance is

around 150Å, and the linker region

sequesters two complex modules, which

makes it possible to solve the complex

structure within condensed phase by single

particle cryo-EM.

2D membrane system

Signal transduction between cells cannot skip

over membranes. Supported lipid bilayer has

been a popular working model to mimic cell

membrane in vitro for years (60). People

have noticed large number of membrane

proteins such as adhesion molecules,

receptors and channels that are required to be

assembled/enriched/clustered together to

transmit signals, but conventional protein-

protein interactions can hardly elucidate the

coupling principle until phase separation

came into sights. Reconstitution of

transmembrane protein clustering on

supported lipid bilayer is important for

studying the mechanism of formation and the

functional consequences of these

microclusters (Fig. 4A). A recent review by

Lindsay et al has summarized the

significance of LLPS in transmembrane

signaling (61). Here we discuss about some

applications and their caveats when dealing

with supported lipid bilayer in phase

separation systems.

To visualize the clustering of membrane

proteins on supported lipid bilayer, either

Total Internal Reflection Fluorescence (TIRF)

or confocal microscopy can be performed

considering the membrane thickness is much

below the optical microscopy resolution.

Traditional imaging methods like

colocalization, fusion, dispersion, and FRAP

can also be conducted to describe the

ensemble behaviors of proteins on supported

lipid bilayer. In addition, all the materials are

restricted to a single membrane sheet which

theoretically accounts for all the signal

sources, making it more convenient for

quantification. Firstly, fluorescence

intensity-based quantification methods as

described for analysis of droplets in 3D

solution is applicable. Same fluorophore can

be conjugated to either lipid or protein, the

absolute number of lipids can be calculated

by the known coating surface area and lipid

head group size. A standard curve of

fluorescence intensity with respect to the

number of fluorophores can then be plotted.

Assuming the illumination property of a

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fluorophore remains the same no matter

being conjugated to lipid or to protein,

protein density can thus be converted by

referring to the standard curve generated

from the labeled lipid (41,45). An alternative

approach is to take advantage of the bilayer

membrane to perform single molecule

counting. The protein of interest is labeled

with two different fluorophores, where one is

sparse enough for single molecule counting

and the other for experiments. One can then

calculate the molecular number from the

known concentration ratio of sparse labeled

fraction before coating (42,62).

Furthermore, the confinement of

membrane protein on supported lipid bilayer

allows one to trace proteins at single

molecular level (Fig. 4B). STORM provides

a method to delineate behaviors of membrane

localized protein microclusters (45).

Distribution of molecules is directly counted

(i.e. bypassing the fluorescence intensity

conversion in bulk imaging experiments),

and the concentration ratio can be extracted.

The dynamics of individual molecules is

directly evaluated by tracking their

individual movement trajectories instead of

depending on the overall fluorescence

recovery as in FRAP analysis. Trajectories of

single molecules over time can be

categorized, and it has been shown that

molecules within condensed phase move

significantly slower than their counterparts in

dilute phase (Fig. 4B). Super resolution

imaging is a powerful technique for

illustrating features of individual molecules,

but deliberations need to be taken for the

compatibility of the two systems. For

example, the imaging buffer for STORM

experiments contains a thiol compound to

enable photo-switching. However, when a

His-tagged protein attaches to the membrane

via interaction with DGS-NTA-Ni2+ lipid to

mimic its membrane localization, reducing

reagent can interfere with membrane

attachment. Fortunately, this system is

tolerant to sub-dosage of 2-mercaptoethanol

to some extent while sufficient amount of

photo-switching can still be achieved (45).

Another concern is the requirement of

oxygen scavenger system that consists of

glucose, glucose oxidase, and catalase. High

concentration of glucose (~10%) may affect

the ability of the molecular components to

phase separate, although the influence is case

by case and rigorous controls are required. In

addition, the intermediate product, hydrogen

peroxide, will oxidize and destroy the lipid

membrane if failed to be eliminated by

catalase. Therefore, the relative

stoichiometry of imaging buffer components

and duration of imaging time are crucial to

maintain a reduced environment at all times.

Open questions

The application of 2D supported lipid

bilayer system for characterization of LLPS

is still at the initial stage and in the ascendant.

Many important questions remain to be

considered to promote the development of

this system, and in return, to facilitate the

thorough comprehension of this field. In

current systems, the coating of membrane

proteins mainly relies on NTA-Ni2+-His

interaction for simplicity. Clustering of

membrane proteins, however, will drag

synchronized movement and clustering of

NTA-lipids, which may affect the overall

membrane properties. Besides, the protein

coating efficiency is dependent on the

proportion of NTA-lipid (DGS-NTA), but the

percentage of DGS-NTA itself will affect

membrane fluidity as indicated by FRAP

analysis (63). Continuous efforts are thus

demanded to overcome this problem. For

example, transmembrane proteins might be

inserted into supported lipid bilayer

independent of NTA-Ni2+-His interaction.

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The lipid components we are looking at in

current studies are way too simplified

comparing to those in natural conditions.

Importantly, lipid compositions can change

over time and in response to cellular

activities, and protein-lipid interactions are

often involved in signal transduction and

regulation. In addition, lipid itself can

undergo phase separation, which represents

another fascinating research field (64). What

happens if protein phase separation comes

across lipid phase separation? Membrane

bilayers constituting more close-to-

physiology lipid compositions should

certainly be taken into consideration over the

long haul. The concept of “membraneless

compartment” is gradually becoming a

consensus, and studies performed with

supported lipid bilayer increasingly uncover

the relationship between protein condensates

in solution and protein clusters on lipid

membranes. Early evidences have already

shown a direct connection between synaptic

vesicle pool and its buffering surrounding−

synapsin phase separation in presynaptic

termini (56). In the future, it will be

interesting to study how membraneless and

membrane-bound compartments are coupled

using a combination of 3D solution and 2D

membrane systems.

Functional implications

We have so far discussed the molecular

mechanisms that drive phase separation and

how to characterize LLPS in solution and on

lipid bilayers in vitro. Studies on in vitro

reconstitution systems shed light on the

biological significance of phase separation.

In this section, we propose a few potential

functional implications of having non-

membrane enclosed biological condensates

in cells.

Compartmentalization without physical

barriers

Membrane-mediated molecular

confinements guarantee specific

proteins/nuclei acids subcellular localization

thus allowing distinct functions of each

organelle. However, membrane-bound

organelles with limited types are insufficient

to support diverse cellular processes with

multiple functions. Cytoplasm should be

further segregated to control each unique

chemical reaction without potential

disturbances. Functional proteins are often

found to have their preferential sites in a cell

with a sharp concentration gradient to the

neighboring environment. Phase separation

among different biomacromolecules

facilitates spontaneous formation of different

subcellular compartments without the help of

lipid membranes. Since phase separation is

driven by intrinsic properties of an exact

protein and its binding partners, such

compartmentalization can be highly specific

to its inner components. The transition is

achieved in a membrane-independent manner,

therefore cells can simultaneously

condensate different materials into various

compartments, each with defined

constitution and function (Fig. 5). Moreover,

forming membraneless organelles would be

“energy friendly” to cells because lipid

biogenesis and membrane identity

maintenance consume a huge amount of

energy. Phase separation by molecules under

physiological conditions is a natural process

with no demands for extra energy, thus cells

do not need to actively deliver materials

towards each condensate against a huge

concentration gradient. The membraneless

and liquid-like properties allow a newly

formed condensate to fuse with another to

enlarge its size, a process which could also be

energy costly if every condensate is enclosed

by membranes (Fig. 5).

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Achieve high local concentrations for

molecular interactions and rapid chemical

reactions

Self-condensation is one of the key

features and probably also the most

important function of phase separation.

Compared to macromolecular complexes

formed by traditional interaction mode, a

demixed phase enables molecules to be

massively enriched into a restricted

subcellular region and subsequently to

increase their local concentrations by

hundreds of folds. This massive increase in

concentration brings at least two non-

negligible changes towards materials inside

the condensates. First, for scaffolding

proteins involved in assembly of the entire

architecture, weak interactions between

molecules, which usually is almost

undetectable in aqueous solution, can get

dramatically amplified and contribute to the

properties of biological condensates (65,66).

That could explain why sometimes a single

amino acid substitution on a given protein,

which hardly changes its behavior in

homogenous solution, might severely

influence its ability to phase separate. Those

previously identified weak interactions

should also be re-evaluated taken into the

consideration of phase separation, because

they may no longer be nonspecific or without

any functional implications. Secondly, higher

local concentration of enzymes enriched in

condensates might show altered activities or

kinetics during chemical reactions. If a given

enzyme gets concentrated into condensed

phase with an open conformation, the active

recruitment or exclusion of its substrate

determines whether a chemical reaction

would get promoted or inhibited (Fig. 5).

CaMKII is the most abundant enzyme in

synapses, colocalizes with its numerous

substrates in PSD and exhibits neuronal

activity-dependent translocation into synapse

from dendritic shaft (67). Upon kinase

activation, one might foresee enhanced

phosphorylation of CaMKII substrates to

activate downstream signaling pathways.

Actin polymerization provides another

example of how phase separation can

promote reaction kinetics. In nephrin and

LAT systems, the amount of actin assembly

is dramatically upregulated when the

signaling components undergo phase

separation (discussed above). It has recently

been demonstrated that the increased

membrane dwell time of N-WASP, in the

condensed phase, promotes its association

with the Arp2/3 complex and subsequently

the actin polymerization rate compared to

homogenous solution state (10,41,68,69).

The formation of astral microtubules from

centrosome is also promoted when tubulin

monomers become massively enriched into a

reconstituted centrosome condensate by

SPD5 (66). Stress granules provide a

contrasting example where protein

translation is sequestered by actively

“squeezing” mRNAs and some of the

translational machineries from cytoplasm

into a densely packed condensates (69).

Future experiments should be focused on

both accurately measuring the enzyme

kinetics and systemically proposing reaction

theories in condensed phases.

Allow fast changes of molecules upon

signaling

Membrane-enclosed structure shows

reduced molecular dynamics because its

inner materials are completely segregated by

lipid bilayers. Condensates formed by phase

separation can confine molecules to a given

region but meanwhile allow them to freely

exchange with their counterparts in the

surrounding solution. This type of molecular

dynamics provides a unique feature of phase

separation-mediated condensation—to

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rapidly rearrange its interior constituents in

response to different stimuli. Compositional

reorganization within a particular

compartment can be accomplished by

selectively altering the behavior of a given

protein with covalent modifications that

favor or disfavor its local environment. For

example, Synapsin undergoes phase

separation by itself and further cluster

synaptic vesicles (56). This condensation can

be dissolved upon Synapsin phosphorylation

by CaMKII. Arginine methylation on FUS

protein did not affect its phase separation

ability, but dramatically decreased the

hardness of FUS droplets, indicating that

post-translational modification could also

modulate the material properties of a given

condensate (34). Phase separation might also

undergo an overall weakening when the key

organizer is depleted or competed off by

other regulatory molecules. The dispersion of

reconstituted PSD phase droplets by an

alternatively spliced form of Homer1

provides another good example of biological

condensate regulation (44). Since

multivalent intermolecular interactions (both

strong and weak) are the driving force of

phase separation, one could imagine that the

condensation process can be extremely

sensitive to changes in the outside

environment including salt concentration, pH,

temperature, redox conditions etc. Tuning

biomolecular interactions might bring huge

influences on a condensed phase, making

each condensed system a perfect biosensor

that enables cell to recognize various signals

and make rapid responses to them.

Sub-segregation via phase-in-phase, phase-

to-phase, or surface coating

Organelles with multiple membrane

layers are not commonly used in living cells.

Mitochondria and chloroplasts are the only

two known systems with double layers of

lipid membrane which allow their inner

materials to be further segregated to facilitate

multistep reactions during respiration and

photosynthesis. Sub-segregations within an

organelle can provide new isolated regions

with distinct functions but meanwhile allow

each segregated part to communicate with

each other. This smart design might also be

adopted by membraneless condensates to

support themselves with multiple functions.

Sub-segregation can happen when multiple

proteins co-cluster into the same condensates

with one of them forming a smaller droplet

and being totally embedded among the other,

a phenomenon termed as phase-in-phase (Fig.

5). For example, three subcompartments

(NPM1, FIB1 and POLR1E) of nucleoli in

Xenopus laevis form distinct and immiscible

liquid phases where FIB1 and POLR1E

condensed into smaller-sized puncta inside

single NPM1 condensate (57). A completely

buried phase is isolated by outer layer

proteins thus preventing potential dynamic

exchange. At the same time, protein

concentrations would further increase as the

total volume of a droplet gets smaller, which

might vastly speed up reactions inside the

condensate. When sizes of two sub-

segregation become similar, a layer-to-layer

structure could form with two droplets

sharing a common interface but each exposed

to the outside environment. A condensation

organized in a phase-to-phase pattern can

generate multiple functional interfaces for

biomolecular interactions and signaling

transductions with specific orientations (Fig.

5). During germline development in C.

elegans, two P granule proteins ZNFX-1 and

WAGO-4 become phase separated from P

granule to form an independent liquid phase.

This newly formed phase further assembles

into tri-condensation with P granule and

Mutator foci in a phase-to-phase manner to

spatiotemporally regulate epigenetic

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inheritance during development (70). The

detailed molecular mechanisms for phase-

in/to-phase is still not well understood, and it

is believed that both the interaction among

inner materials and the surface tension of

individual droplets may govern the sub-

segregation process. Surface coating is

another unique type of sub-segregation when

some molecules only localize to the surface

of a transitioned phase (Fig. 5). Recruitment

of particular molecules to droplet surface

might change the surface properties of a

given condensate and offer it with new

functions. In addition, biomolecules, even

without the help of transmembrane or

membrane-binding domains, can be confined

into a 2D system, which dramatically alters

their activities. This might be achieved when

a given protein has two featured surfaces, one

of which favors the inner environment of a

condensed phase but the other disfavors and

gets excluded. Thus, surface coating is

regarded as an equilibrium between protein

attraction and exclusion from materials

inside the condensate. Surface coating to a

selected phase droplet may also affect its

material properties. Molecules on the

condensate surface can regulate dynamic

exchange of its inner materials, influence free

diffusion of small molecules, and even alter

fusion process of droplets. A recent study

reported that EPG-2, a scaffold protein in C.

elegans P granule, can specifically decorate

the surface of SEPA/PGL-1/-3 droplets and

modulate the condensate properties (71).

Direct communications between

membraneless and membrane-bound

organelles

Membraneless organelles formed by

phase separation could communicate with

membrane-bound organelles via direct

interactions (Fig. 5). Such communication

might help to specify the localization of

membrane-bound organelles, to reorganize

protein distributions on membrane surface

and to introduce new functions to organelles.

TIS11B, an RNA-binding protein, for

example, forms membraneless granules that

directly attaches to ER (72). TIS granules

specifically retain certain mRNAs and

exclude others to enable accurate protein

translation in ER. RNA granule is also

observed to associate with late endosomes

residing close to mitochondria in neuronal

axons to regulate local synthesis of axonal

proteins (73).

Phase separation and evolution

Previous subsections discuss the

functional implications of phase separation in

terms of offering a living cell with multiple

functions. Here we postulate on possible

biological importance of phase separation

from the angle of life evolution. It is hard for

one to imagine that the earliest form of life

directly starts with membrane-bound

organelles as lipids are not typical

information carriers like nuclei acids. In

addition, the biogenesis of lipids depends on

other molecules with catalytic activities, such

as protein or RNA. The origin of life is

believed to depend on RNA because it carries

the genetic information and possesses

enzymatic activity which many allow them to

self-reproduce. RNA is so unique as it forms

a long chain with multiple binding sites for

other RNAs or proteins, which might also

explain why it could always easily undergo

LLPS with different RBPs or even by itself.

Compartmentalization mediated by phase

separation may reveal how proteins and

nuclei acids assemble into condensed

bioreactors in ocean before the emergence of

lipid membranes. Indeed, membraneless

condensation is observed not only in higher

animals but also in ancient cyanobacteria,

indicating a common and conserved

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biological process that could be shared by all

the living creatures on earth (46). We refer

readers to several recent reviews (6,74,75)

that also discussed about the emerge of LLPS

process during the evolution.

Conclusions and perspective

In cells, biomaterials can be organized

into membrane-bound or membraneless

compartments. Significant progresses have

been made over the past decade in

understanding the mechanisms underlying

the formation and organization of non-

membrane enclosed organelles. Many of

these systems are driven by LLPS via which

collections of molecules demix from the bulk

solution/cytoplasm to form biological

condensates. Modular domain proteins and

intrinsic disorder containing proteins exhibit

multivalent intermolecular interactions either

via specific, high affinity interactions or

weak adhesions that drive phase separation.

Analysis of features of molecules involved in

LLPS has started to reveal sequence

determinants in intrinsically disordered

regions that promote phase separation.

Although our understanding is still

rudimentary, it is clear that certain sequence

patterns are heavily involved and can

determine the material properties of a

condensate. A large collection of methods has

been developed to study the dynamics,

composition and physical properties of

condensed droplets in vitro. We still need

more quantitative assays, measurements and

descriptions for future phase separation

studies. For instance, theoretical work is

required for understanding the underlying

physical and chemical principles of LLPS.

Bioengineering tools may be designed to

precisely control phase separations in vitro or

in vivo. Another appealing, yet very

challenging, idea is to reveal the atomic

details of the condensed droplets. In

particular, could there be a single structure

that might be “solved”? Cryo-EM studies

have been conducted on some condensates

trying to answer this question, although little

success has been achieved so far. This is

somewhat expected since many of the phase

separations are contributed by intrinsically

disordered elements. It is difficult to imagine

how the multivalent interactions might be

restricted to oligomers of homogenously

distributed sizes. Nevertheless, we cannot

rule out the possibility that a core structure of

defined stoichiometry and conformation

might exist among other flexible and

heterogeneous structures, especially where

the condensate formation is driven by

modular domain interactions. To “solve” the

atomic structure of phase condensates might

be too optimistic at current stages, but it is

possible that we might reveal the molecular

organizations within the condensates using

cryo-ET and cryo-EM techniques. Is there a

layered organization within the phase

droplets? How do molecules assemble into

supramolecular complexes that phase

separate from the bulk solution? Results from

in vitro characterizations of phase droplets

would provide insights into how the

macroscopic properties of condensates might

contribute to their biological functions in

cells. There remains much to answer about

the biological condensates. How is the

enzyme kinetics regulated in condensed

phase? Recent studies using nephrin and

LAT systems have shown that increased

dwell times of enzymes in condensed phase

lead to faster reaction rates (62,68). Is this a

general mechanism for other molecular

systems? Compared to membrane-bound

organelles, LLPS-mediated membraneless

structures have their own advantages; but it

also brings many potential problems. For

example, how to achieve specificity in

organization? How to prevent unwanted

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fusion or mixing without a membrane barrier?

How does sub-segregation happen inside an

organelle and how to maintain this specific

pattern? Is this physiologically relevant and

functionally regulated? Will the concept of

phase separation help us to re-examine many

diseases that are hard to be explained by

physical chemistry principles of dilute

solution systems? For instance, alteration in

the material properties of many neuronal

protein condensates might contribute to

neurodegenerative diseases. The

concentration-dependence of phase

separation might help explain the dosage

sensitivity of SynGAP, a negative regulatory

protein of PSD assembly, in psychiatric

diseases. Answers to these questions will

provide us with in-depth insights into

mechanisms underlying the formation and

regulation of biological condensates in cells

and to understand how nature evolves this

type of compartmentalization in life. In the

end, although the list of non-membrane

bound organelles formed by LLPS continues

to expand, researchers should always ask

themselves— is the LLPS-driven protein

condensation observed in vitro biologically

relevant? If so, what is its contribution to

cellular functions? Can the in vivo

observations be explained by mechanisms

other than liquid phase separation (76)?

Tailored experiments need to be designed in

order to distinguish between these

possibilities. Nonetheless, it is assured that

LLPS-mediated biological condensate

formation is an emerging life science

research field with numerous exciting

opportunities.

Acknowledgments

Work in our laboratory is supported by grants from RGC of Hong Kong (AoE-M09-12 and

C6004-17G) and a grant from Simons Foundation for Autism Research (510178). ZF is

supported by Mandatory Provident Fund Scheme and is a Junior Fellow of IAS at HKUST. MZ

is a Kerry Holdings Professor of Science and a Senior Fellow of IAS at HKUST.

Competing interests

The other authors declare that no competing interests exist.

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FIGURE LEGENDS

Figure 1. Types of multivalent interactions driven by intrinsically disordered elements in

liquid-liquid phase separation (LLPS) systems.

(A) Phase diagram constructed by varying protein concentration and storage conditions such as

buffer reagents and temperature. Solid line depicts the boundary at which molecules reach their

solubility limit and become immiscible with the surrounding solution. Grey box highlights

confocal image showing the homogeneous solution state of NR2B C-terminal tail (labeled with

Alexa Cy3) in the absence of PSD scaffold proteins. Conditions within the spinodal curve

(indicated as dashed line) are where spinodal decomposition occurs. Example of fluorescence

image, highlighted by green box, showing that the membrane tethered NR2B tail (labeled with

Alexa Cy5) formed clusters on supported lipid bilayers upon the addition of major PSD scaffold

proteins. Phase separation is only observed in the presence of a nucleation process when

conditions lie in between the binodal (indicated as solid line) and spinodal curves.

Representative image, highlighted by yellow box, showing the clustered state of NR2B tail

(labeled with Alexa Cy3) in 3D solution in the presence of major PSD scaffold proteins

(adapted from Ref. 45). Scale bar, 10 μm.

(B) Aromatic residues in intrinsic disorder containing proteins are involved in pi-pi or cation-

pi interactions with positively charged residues such as Arg and Lys. RGG repeats are

frequently found in low complexity regions (LCR).

(C) Patterned charge distributions to facilitate electrostatic interactions between oppositely

charged residues.

(D) Secondary structural elements are involved in multivalent intermolecular interactions, such

as the kinked cross β sheets formed by a segment of FUS LCR (PDB code:6BWZ).

Figure 2. Types of multivalent interactions driven by modular domains in LLPS systems.

(A) Interaction network of N-WASP, nephrin and NCK.

(B) Schematic representations showing the network of multivalent interactions involving major

PSD proteins. Solid line indicates direct modular domain interactions. Dashed line indicates

indirect recruitment of actin filaments via Shank3 and Homer proteins.

(C) Schematic interaction network of presynaptic active zone proteins RIM and RIM-binding

protein together with the cytoplasmic tail of the N-type voltage gated Ca2+ channel (NCav).

(D) RNA binding proteins, hnRNPA1 for example, binds to RNA triplets via RNA recognition

motifs (RRM). Multiple RRM-RNA interactions, albeit of low affinities, together provide

multivalency to drive phase separation.

Figure 3. Techniques for characterizing of condensed phase formed in 3D solution.

(A) Differential interference contrast (DIC) (left image) coupled with fluorescence imaging

(middle and right images) of the phase droplets, multiple labeling of different components

demonstrate their colocalization.

(B-D) Dynamic properties of the condensed phase.

(E) Fluorescence intensity based absolute concentration estimation (adapted from (44,45)). A

standard curve of fluorescence intensity to dye concentration is initially generated for

calibration. Z direction scanning is performed to determine the proper focal plane for

concentration estimation. At each Z stack, the fluorescence intensity distribution is plotted.

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Within the Z dimension of selected droplets, average fluorescence intensities are then compared

across different layers. In a given system the fluorescence intensity is constant regardless of the

droplet size, and therefore the absolute protein concentration within a condensed phase can be

calculated from the standard curve.

Figure 4. Condensed phase formed on 2D supported lipid bilayer.

(A) Schematic diagram of microdomain formation on 2D supported lipid bilayer. Membrane

proteins homogeneously distribute on supported lipid bilayer via the tethering of His-tag to

Ni2+-NTA decorated lipids. Protein clusters are observed on lipid bilayers after the addition of

other components to drive phase separation.

(B) Stochastic optical reconstruction microscopy (STORM) analysis of membrane proteins, the

cytoplasmic tail of NCav as an example, on supported lipid bilayer (adapted form (45)). Image

captured under total internal reflection fluorescence (TIRF) microscopy mode first sketches the

contours of the condensed phase, which turns out to perfectly overlap with the image

reconstructed from STORM analysis. Trajectories of individual molecules are followed by

single molecular tracking assay, both inside and outside the condensed phase. Direction of

movement is marked by gradient color from black to red.

Figure 5. Biological functions of LLPS-mediated membraneless compartments.

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Zhe Feng, Xuedong Chen, Xiandeng Wu and Mingjie Zhanganalytical methods, and physiological implications

Formation of biological condensates via phase separation: Characteristics,

published online August 23, 2019J. Biol. Chem. 

  10.1074/jbc.REV119.007895Access the most updated version of this article at doi:

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